1. Field of the Invention
The present invention relates to a fluorescence polarization measurement system in a lab-on-a-chip and, more particularly, to a method and an apparatus for measuring fluorescence polarization FP in a lab-on-a-chip that analyze quantitatively interactions between minute amounts of biomolecules using fluorescence polarization, and a method for detecting a substance that induces or inhibits formation of a complex of biomolecules using the method for measuring fluorescence polarization in a lab-on-chip.
2. Description of Related Art
A fluorometry that measures fluorescence provides one of the important methods in bioassay techniques. The fluorometry includes fluorescence intensity, fluorescence fluctuation spectroscopy, fluorescence imaging, fluorescence resonance energy transfer (FRET), and fluorescence polarization FP. Due to its sensitive nature, the fluorometry has been continuously substituted for assay methods using radioactive isotopes.
Among these, fluorescence polarization FP, the concept of which was introduced by Perrin, is directed to a method of measuring the time-averaged rotation motion of a fluorescent molecule. Therefore, the measurement of fluorescence polarization FP is based on the principle that a fluorescent molecule generates FP when it does not rotate in excited state, whereas a fluorescent molecule emits fluorescence in all planes, resulting in loss of FP, when it rotates freely by Brownian motion in the excited state.
The method for measuring fluorescence polarization FP has been acknowledged as a powerful and unique method in analyzing interactions between molecules. Fluorescence polarization FP is generally inversely proportional to rotation time of molecules and influenced by absolute temperature, and molecular viscosity and volume. Such method provides a direct assay that does not require a solid phase separation and is capable of measuring interactions between fluorescently labeled biomaterials and other biomolecules with high sensitivity, owing to the relation of FP to changes in molecular size. That is, the method analyzes interactions between biomaterials based on the principle that when the net molecular volume increases due to the binding of small fluorescently labeled biomaterials with other biomolecules, fluorescence polarization FP increases, whereas, when the molecular volume decreases due to biomolecules′ separation, FP also decreases.
A system for measuring fluorescence polarization FP is configured generally in a manner that when monochromatic light passing through a polarizer excites fluorescent molecules in a sample tube, only molecules oriented toward the same polarized plane as the irradiating light absorb the light to be excited. Then, the light emitted from the excited molecules is measured in both vertical and horizontal planes.
The values of fluorescence polarization FP that express how much the molecules rotate during excitation and emission are calculated according to:FP=(IVV−IVHG)/(IVV+IVHG)
wherein IVV represents a vertical fluorescence intensity; IVH denotes a horizontal fluorescence intensity; and G (G-factor; IVV/IVH) is an empirical constant that corrects for the polarization bias introduced by the optics and the detection system. To apply the method for measuring fluorescence polarization FP to mass analyses, scanning methods based on the principle of fluorescence microscopy are generally used for the samples processed in well plates. The amount of the sample used is about 100 μL in case of a 96-well plate and about 40 μL in case of a 384-well plate.
A system for measuring fluorescence polarization FP used on capillary electrophoresis has been reported to involve separation of a biomolecular complex via capillary electrophoresis and collection of the separated complex using a cuvette cell to measure the fluorescence intensities in both vertical and horizontal planes, thus obtaining fluorescence polarization. Since the system described above comprises two steps of separating and measuring and uses a relatively large cuvette cell (0.2 mm×0.2 mm), it has a drawback in collecting a considerable amount of separated biomolecules for the large cuvette cell [Anal. Chem. 72(2000) pp. 5583-5589].
The use of fluorescence polarization FP, on the other hand, has not been extensively explored for applications in lab-on-a-chip devices although it serves as a valuable technique for analyses of biomolecular interactions in conventional well-plate formats and by capillary electrophoresis. Only very recently, biosensing of homogeneous molecular binding by FP detection using a commercial fluorescence spectrometer has been attempted in relatively large plastic microchannels [Anal. Chim. Acta 507 (2004) pp. 123-128].
The system using the above plastic channel mixes an analytic solution with fluorescently labeled antibody/enzyme in a reservoir to form a complex. Then, the complex flows in a plastic channel having a diameter of 300 μm to 500 μm, which is made of poly dimethyl Siloxane PDMS Block. The channel containing samples is irradiated with polarized light, and the emitted fluorescence is measured on a conventional spectrometer to yield fluorescence polarization FP as shown in FIG. 1.
Here, substances mix in the reservoir flow in a disposable plastic channel having a larger diameter of 300 μm to 500 μm to be measured by the conventional fluorescence spectrometer. Although the measurement can be readily carried out, it requires a considerable volume of about 10 μL and also a good many quantity of 10 nmol to 40 nmol of samples. In addition, the use of the disposable plastic channel has some drawbacks in that it is difficult to monitor the reaction of continuously flowing samples, and to measure the fluorescence signals due to large background signals in a miniaturized microchannel.
Meanwhile, a lab-on-a-chip is a kind of biochip integrating various devices for sample preparation, sample injection, reaction, separation, measurement and the like on a substrate having a size of several square centimeters and made of glass, silicon or plastic using a technique of photolithography used in fabricating semiconductors. That is, the lab-on-a-chip means a physical, chemical and biological microprocessor that a laboratory is put on. The lab-on-a-chip includes a microchannel made of plastic, glass, silicon or the like, through which samples of nanoliters or less are tested. Accordingly, various experiments performed in existing laboratories can be executed in such a lab-on-a-chip and it is possible to carry out automated experiments at a high speed, and with high efficiency and low cost using the lab-on-a-chip.
Especially, the lab-on-a-chip has attracted attention as a next-generation diagnostic apparatus as biotechnologies have rapidly developed since the year 2000. Using this chip, only a drop of blood would be enough to diagnose various cancers or measure the number of erythrocytes and leukocytes. In addition, the lab-on-a-chip is a higher value-added product that can expand its application into numerous fields such as stock breeding, environment, etc.
However, a high-throughput analysis system for measuring fluorescence polarization FP in a lab-on-a-chip has not been well known. With this system, it is possible to execute an interaction analysis by fluorescence polarization FP with high sensitivity using a minute amount of a sample.
Various techniques on identification and quantification of the activity of proteolytic enzyme have been developed, including measurements of absorbance or fluorescence liberated in the supernatant of precipitation assay, homogeneous fluorometric assays using fluorescence-quenched, hyperconjugated fluorescein derivatives of protease substrates, and fluorescence polarization FP based assays of fluorescently labeled-protein substrate. The former suffers from the need for careful sampling intervals, control of sample volumes, and separation of labeled hydrolysis products from unhydrolyzed protein, while the latter two appear to provide a rapid and convenient measurement system. Since fluorescence polarization FP is independent of fluorescence intensity and thus more tolerant of fluorescence intensity fluctuations, it is possible to measure the activities of various proteases sensitively by the fluorescence polarization FP based assays.
The existing assay method for the activity of protease is to use a fluorescently labeled peptide substrate for a specific protease. But the method has a problem in that it needs specific substrates for the respective proteases. Accordingly, the assay system for the activities of proteases in a lab-on-a-chip using fluorescence polarization FP and a universal protein substrate, which will be described hereinafter in various embodiments of the present invention, is generally applicable to various proteases.
The inventors of the present invention have tried to develop a system for measuring fluorescence polarization FP in a lab-on-a-chip using a sample of several pmol to several tens of fmol, and completed a method and an apparatus for measuring fluorescence in a lab-on-a-chip of the present invention that analyze quantitatively interactions between biomolecules and fluorescently labeled biomaterials, and a method for detecting a substance that induces or inhibits formation of a complex of biomolecules using the method for measuring fluorescence polarization FP in a lab-on-a-chip.